Multi-layer cell encapsulation for tissue engineering

ABSTRACT

A multi-layered microcapsule has an inner extracellular matrix and an outer shell. The inner extracellular matrix includes a first inner layer of biopolymer and a second intermediate layer of polymer that provides partial immune-protection and holds the first layer in place. The outer shell can form an exoskeleton to provide mechanical stability. Each of the individual layers can be varied to optimize mechanical stability, cell function, and immuno-protection.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. application Ser.No. 09/975,273, filed Oct. 12, 2001, which claims benefit under 35U.S.C. § 119(e) to U.S. application Ser. No. 60/239,259, filed Oct. 12,2000, the disclosure of each of which is hereby incorporated byreference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to cell encapsulation and, more particularly, toencapsulating living cells in a multi-layer polymeric membrane.

2. Description of Related Art

Microcapsules for biological substances are composed of thin,semi-permeable membranes of cellular dimensions. Microcapsules can beprepared of various polymers and their contents can consist of enzymes,cells and other biological materials. Microcapsules are prepared in sucha way as to prevent their contents from leaking out and causing animmunological reaction, but the microcapsules still allow the nutrientsand metabolites to exchange freely. This method has found applicationsprimarily in transplantation of foreign materials in vivo withoutimmunosuppression. One example is microencapsulation of hepatocytes foruse in bio-assisted liver devices (BLAD). The surface-to-volume ratio ofa spherical microcapsule facilitates maximal transport of nutrients,gases, or metabolites exchange across the membrane. In addition,encapsulation of living cells allows better control of themicroenvironment for optimal cellular functions via selection ofsuitable substrate and incorporation of controlled-release features intothe local microenvironment. Other physical characteristics such as masstransport, mechanical and chemical stability can also be configured asdesired without drastically affecting the functions of the living cellsinside the microcapsules.

The commonly used techniques for cell encapsulation are complexcoacervation and interfacial precipitation. Complex coacervationinvolves the electrostatic interaction of two oppositely chargedpolyelectrolytes. At the right matching charge density, the twopoly-ions combine and migrate to form a colloid-rich or water-insolublephase. The molecular weight and chain conformation parameters of thepoly-ions may also play an important role in the complexation process.Interfacial precipitation simply relies on the solidification of adissolved polymer upon contact with an aqueous phase.

One of the most extensively studied cell encapsulation schemes is onethat involves an alginate-gelation complex coacervation method. In thissystem, alginate, a glycuranan extracted from the brown seaweed algae,can be chelated by calcium or other multivalent counter-ions to form agel. These early in vivo results with the alginate-polylysine systemhave not been consistent because of the uncontrolled purity of alginate,and the incorporation of cells into the external membrane. As a result,a 2-step encapsulation was developed to further shield sensitive cellsfrom the extra-capsular environment. The living cells were mixed withsodium alginate and extruded into calcium chloride to form calciumalginate gel droplets. These gel droplets were incorporated into largeralginate gel spheres and then reacted with a poly-amino acid such aspoly-L-lysine to form a semi-permeable membrane. Incubating with sodiumcitrate liquefied the interior to form microcapsules. Unfortunately, theaddition of sodium citrate appears to have affected the functions of thecells. Furthermore, the water-soluble alginate and poly-lysine wereshown to be not particularly biocompatible as individual polymers, othermatrices such as collagen may be better substrates for cellularfunctions than alginate.

To encapsulate living cells in natural matrices such as collagen,interfacial precipitation has been used. In this method, hydroxylethylmethacrylate-methylmethacrylate (HEMA-MMA) solution in dimethylformamide and cell-suspension in collagen or Matrigel were extrudedseparately through two concentrically configured needles into aprecipitating bath containing largely water with a floating layer ofdodecane. Polyacrylates are water insoluble that enhances the in vivostability of the microcapsules. The living cells encapsulated this way(especially with Matrigel) survive well. The interfacial precipitationrequires a more elaborate setup than the complex coacervation to controlthe microcapsule sizes and minimize the contact of cells with organicsolvents.

In U.S. application Ser. No. 09/414,964, filed Oct. 12, 1999, anegatively charged ter-polymer of hydroxyethyl methacrylate-methylmethacrylate-methacrylic acid (HEMA-MMA-MAA) is used to encapsulatecells within a positively charged collagen. The MAA added into theter-polymer enhances the water solubility of the polymer, allowing theentire encapsulation to be carried out in an aqueous environment. Hence,the complex coacervation method is used while a simple setup providesfor easy control of the microcapsule size. The resulting hepatocytemicrocapsules exhibit enhanced cellular functions as well as desirablephysical characteristics for use in bio-artificial liver. Themicrocapsules, however, were mechanically unstable as measured bynano-indentation method. After 4 days of static in vitro culture, themicrocapsules became weak and breakable upon harsh handling. Attempts atimproving the mechanical stability of the microcapsules resulted intradeoffs with immune-barrier/mass transfer efficiencies and cellularfunction.

There remains a need for improved microcapsules that exhibitsatisfactory mechanical stability in combination with improvedimmune-barrier/mass transfer efficiencies and cellular function.

SUMMARY OF THE INVENTION

The present invention, according to one aspect, is directed to amicrocapsule for culturing cells, particularly anchorage-dependentcells. An inner, extra-cellular matrix surrounds the cells. The innerextracellular matrix can be formed from a biopolymer inner layer and abiocompatible synthetic polyelectrolyte outer layer, wherein the innerlayer and the outer layer have charges sufficient to form a complex ofthe biopolymer and the polyelectrolyte. An outer shell of syntheticpolymer surrounds and supports the extracellular matrix. Themicrocapsules are permeable to nutrients necessary to sustain normalmetabolic functions of the cells and to toxins released by the cells.

According to another embodiment, a microcapsule for culturinganchorage-dependent cells comprises an inner extracellular matrixsurrounding the cells and an outer shell surrounding and supporting theextracellular matrix. The outer shell comprises a macro-porousexoskeleton formed by complex coacervation with the extracellularmatrix. The macro-porous exoskeleton preferably includes suchbiocompatible materials as alumina, alumina sol, or chitosan.

According to yet another embodiment, a microcapsule comprises an innerextracellular matrix surrounding living cells, a macro-porousexoskeleton surrounding and supporting the extracellular matrix, and anouter shell of synthetic polymer surrounding the macro-porousexoskeleton.

The microcapsule membrane preferably is permeable to molecules smallerthan or equal to the size of albumin, to nutrients necessary to sustainnormal metabolic functions of the bioactive cells, and to toxinsreleased by the bioactive cells. The microcapsule membrane preferably isimpermeable to immunoglobulins and macrophages.

The multi-layered microcapsule of the present invention systematicallyaddresses all thee major aspects of the micro-encapsulation development:optimal ECM environment for high cell functions, good mechanicalstability, and reliable immune-protection. Most previous efforts havebeen focused on immune-barrier development while keeping cell viabilityonly. In most cases, cell functions were quite poor. For hepatocyteencapsulation, cell functions never exceeded that exhibited by themonolayer culture control. While some other microcapsules do exhibitgood mechanical stability, the cell functions and mass transferproperties have been unsatisfactory. Therefore, the enhancement of cellfunctions due to encapsulation was not fully exploited; the mechanicalstability was weak; and effective immune-barrier could not be ensured.The multi-layered cell encapsulation of the invention advantageouslyallows all three major properties of the microcapsules to besystematically tuned for required applications in tissue engineering.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in more detail withreference to preferred embodiments of the invention, given only by wayof example, and illustrated in the accompanying drawings in which:

FIG. 1 is a confocal micrograph of a Type-II microcapsule, whichillustrates that the second ter-polymer shell covers the cellsprotruding from the inner ter-polymer shell to ensure a more thoroughencapsulation;

FIGS. 2A and 2B are images of a Type-III microcapsule surface:

FIG. 2A is a scanning micrograph of a Type-III microcapsule using 0.02%chitosan as the exoskeleton biomaterial, and

FIG. 2B is a scanning micrograph of a Type-III microcapsule using 6 mMalumina sol-gel as the exoskeleton material, illustrating a macro-porousnetwork formed by the condensation of positively charged aluminaparticles on the exterior of the microcapsule (scale bars represent 10μm);

FIG. 3 is a scanning micrograph of a Type-IV microcapsule illustratingthat the additional thin layer of ter-polymer resulted in a relativelysmooth fourth layer that was sufficient to cover the exoskeleton beneath(scale bars represent 10 μm);

FIG. 4 is a permeability profile of the microcapsules: BSA (1%) wasloaded into the four types of microcapsules and the rate of BSA releasedfrom the microcapsules was measured (Type-I: (∘), Type-II: (□),Type-III: (x), Type-IV: (⋄)); and

FIG. 5 illustrates a urea production profile of microencapsulatedhepatocytes in Type-I (□), Type-II (Δ), Type-III (x), and Type-IV (∘)microcapsules, as well as a monolayer control (▪).

DETAILED DESCRIPTION OF THE INVENTION

A multi-layer microcapsule comprises an extracellular matrix havingbioactive cells attached to a microcapsule membrane. The microcapsulemembrane has a first inner layer of biopolymer, such as cationiccollagen, anionic collagen, anionic esterified hyaluronic acid, oranionic amine-modified hyaluronic acid, and a second intermediate layerof polyelectrolyte synthetic polymer. As described herein, themicrocapsule also has an outer shell to improve mechanical stability.The microcapsule may include additional layers, such as a fourthoutermost layer of polyelectrolyte synthetic polymer. The layers can beindividually tailored to meet the needs of a particular application.

As used herein, “Type-I microcapsule” refers to cells within abiopolymer, such as a positively charged collagen, encapsulated with apolyelectrolyte synthetic polymer, such as a negatively chargedter-polymer of hydroxyethyl methacrylate-methyl methacrylate-methacrylicacid (HEMA-MMA-MAA). Such microcapsules are described in U.S.application Ser. No. 09/414,964, filed Oct. 12, 1999, the disclosure ofwhich hereby is incorporated by reference.

“Type-II microcapsule,” as used herein, refers to a microcapsuleprepared by re-encapsulating a Type-I microcapsule in biopolymer- andpolyelectrolyte synthetic polymer solutions.

“Type-III microcapsule,” as used herein, refers to a microcapsuleprepared by re-suspending a Type-I microcapsule in an exoskeletonmaterial to form a macro-porous network.

“Type-IV microcapsule,” as used herein, refers to a microcapsuleprepared by re-encapsulating a Type-III microcapsule in apolyelectrolyte synthetic polymer solution.

Both naturally-occurring and modified biopolymers are suitable for useas biopolymers in the practice of the invention, as are both cationicand anionic biopolymers. In general, any commonly used substrates incell studies can be used, non-limiting examples of which includecollagen, cationic collagen, anionic collagen, anionic esterifiedhyaluronic acid, anionic amine-modified hyaluronic acid, fibronectin,and laminin. The biopolymers preferably are water-soluble and most oftenhave a molecular weight of at least 20,000, preferably at least 75,000,more preferably at least 125,000, even more preferably at least 200,000,and yet even more preferably at least 250,000.

Whereas collagen has been used to encapsulate drugs, it has not foundwidespread use for encapsulating cells because, at neutral pH, there isinsufficient charge density to form an encapsulating membrane. However,collagen modified to raise its pKi to at least about 9 is sufficientlypositively charged at physiological pH to be complexed withoppositely-charged synthetic polyelectrolytes to form a coherentmembrane. Collagen can be modified to form a more strongly basic polymerby converting the primary amino groups to tertiary amine groups or byesterification.

Anionic biopolymeric materials, such as hyaluronic acid (HA) andmodified HA (esterified HA or amine-modified HA) are useful in theinvention. In general, anionic biopolymers suitable for the practice ofthis invention will have a charge density of at least about 20%,preferably at least about 30%, and even more preferably at least about50%. HA that is totally or partially esterified or reacted with aprimary amine to render it less water-soluble will form a strongercomplex with the polycationic outer layer than HA itself.

Preferred biopolymers for forming the inner layer of the encapsulatingmembrane are modified HA and modified collagen. Esterified collagen isparticularly preferred as the inner layer. In general, the inner layer,though water-soluble, will be slightly hydrophobic.

Esterification or reaction to form tertiary amine groups on thebiopolymer may be accomplished by reaction of the biopolymer with a widevariety of aliphatic reactants containing as many as about 18 carbonatoms in their chain. Such reactants include, inter alia, alcohols,primary amines and alcohol amines. Preferred reactants contain about 8carbon atoms or less. For some purposes, use of reactants having only 2or 3 carbon atoms may be preferred. Typical alcohols include methanol,ethanol, butanol and higher alcohols, whereas typical primary aminesinclude methylamine, ethylamine and higher amines. Reactants with bothalcohol and amine groups also can be used, such as ethanolamine.Reactants should be chosen so as to not impair the viability of thecells.

The outer layer of the membrane comprises a biocompatible syntheticpolyelectrolyte having a charge opposite that of the biopolymer. Thus,when the biopolymer is polycationic (e.g., modified collagen), thesynthetic polyelectrolyte used in the outer layer is polyanionic.Conversely, when the biopolymer is polyanionic (e.g., HA, modified HA,etc.), the synthetic polyelectrolyte used in the outer layer ispolycationic. Suitable outer layer synthetic polyelectrolytes form acomplex with the oppositely-charged biopolymer to form a membrane by thecomplex coacervation process and impart stability to the encapsulate.The charge density of the synthetic polymer typically will be from about0.1% to about 20%, preferably is at least about 1%, and even morepreferably is at least about 3%. Like the biopolymers, the syntheticpolyelectrolytes preferably have a molecular weight of at least 20,000,preferably at least 75,000, more preferably at least 125,000, even morepreferably at least 200,000, and yet even more preferably at least250,000.

The biocompatible synthetic polyelectrolyte layer that is capable offorming, with the biopolymer of the inner layer, a membrane which allowsenvironmentally-sensitive living cells, such as hepatocyte cells, toremain viable and, at the same time, protects the cells againstimmunological rejection by the host. A preferred class of biocompatiblesynthetic polyelectrolytes is acrylate polymers. Such polymers includeacrylate polymers, copolymers and ter-polymers such as poly(acrylicacid), poly(methacrylic acid), poly(methacrylate), poly(methylmethacrylate), and acrylate copolymers and ter-polymers of acrylic acid,methacrylic acid, methacrylates, methyl methacrylates, hydroxyethylmethacrylic such as 2-hydroxyethyl methacrylate, hydroxypropyl-acrylateand the like, and blends thereof. Poly(dimethylaminoethyl methacrylate)(DMAEMA) and copolymers and ter-polymers of dimethylaminoethylmethacrylate with 2-hydroxyethyl methacrylate and/orhydroxypropylacrylate and methacrylate and/or methyl methacrylate arepreferred cationic synthetic polymers. Copolymers or ter-polymers ofacrylic acid and/or methacrylic acid with 2-hydroxyethyl methacrylicand/or hydroxypropylacrylate and methacrylate and/or methyl methacrylateare preferred anionic synthetic polymers. Each has exhibitedbiocompatibility when used in other biomaterials.

A preferred biocompatible synthetic polyelectrolyte outer layer is anacrylate ter-polymer of methacrylic acid (MAA), hydroxyethylmethacrylate (HEMA), and methyl methacrylate (MMA). The ter-polymerpreferably comprises from about 10 mol % to about 30 mol %, morepreferably from about 15 mol % to about 25 mol % MAA, from about 10 mol% to about 40 mol %, more preferably from about 20 mol % to about 30 mol% HEMA, and from about 20 mol % to about 60 mol %, more preferably fromabout 45 mol % to about 55 mol % MMA. In a preferred embodiment of thepresent invention, the ter-polymer is formed by polymerizing MAA, HEMA,and MMA monomers in about a 1:1:2 molar ratio.

The membrane of the encapsulated cell is selectively permeable. Thecells encapsulated in accordance with the invention remain viablebecause the membrane is permeable to nutrients and other materialsnecessary to support the normal metabolic functions of the cells. Thus,ionic materials and oxygen, for example, pass through the membrane. Themembrane also is permeable to products of the cells, such as hormones,and to metabolic byproducts. Thus, material produced by the cell canpass through the membrane from the interior of the microcapsule. In thisway, material produced by the encapsulated cell can be introduced intothe blood of a host, or can be introduced into a culture medium in whichencapsulated cells are placed.

The membrane permeability essentially precludes entry ofimmunoglobulins, macrophages, and other immune system agents that causerejection of cells by the host's immune system. According to a preferredembodiment of the invention, the membrane is impermeable to moleculesgreater than about 100 kDa, and preferably is impermeable to moleculesgreater than about 71 kDa. According to another preferred embodiment ofthe invention, the membrane is permeable to molecules greater than about60 kDa and impermeable to molecules greater than about 150 kDa.

The composition of the outer layer can be modified to adjust thepermeability and transport properties of the membrane. As an example,the permeability of the membrane to typically polar compounds found inbiological systems can be increased by incorporating a hydrophiliccopolymer, such as poly(2-hydroxyethyl methacrylate) (HEMA) or otherhydroxy-containing acrylates, into the polyelectrolyte which forms theouter layer of the membrane. Increasing hydrophobicity ofpolyelectrolytes tends to cause decreased permeability.

In the preferred MAA/HEMA/MMA ter-polymer, HEMA provides hydrophilicityto render the ter-polymer water-soluble so that the entire encapsulationcan be performed in the physiological aqueous buffer without the needfor an organic solvent. MMA imparts mechanical strength, toughness, andelasticity to the microcapsules. MAA provides a negative charge tointeract with a positively-charged inner layer. The inner layerpreferably is an esterified collagen with net positive charge. Thebalance between the two charged polymers determines the physicalcharacteristics of the microcapsules. Using a 10% ter-polymer and 1.5mg/ml of modified collagen, for example, microcapsules can be formedhaving a thin outer layer (˜21 μm) and a semi-gel-like inner layer thatminimizes impedance to mass transport across the membrane but remainstable as microcapsules for days. The semi-gel-like inner collagen layeris able to provide a “loose” extracellular matrix configuration thatmimics the in vivo situation, therefore allowing the microcapsule tomaintain higher levels of cell function. These characteristics of themicrocapsules that satisfy most requirements for a bioartificialliver-assisted device (BLAD) were achieved through optimization ofseveral parameters.

The permeability of the membrane also can be adjusted by selection ofmolecular weight or structure of the outer layer so as to precludemolecules having a preselected molecular weight or structure frompassing through the membrane. As the molecular weight of thepolyelectrolyte is increased, the membrane tends to be more permeable.Larger differences in charge densities between the inner biopolymer andthe outer polyelectrolyte also tend to make the membrane more permeable.The mechanical stability of the membrane can be improved by increasingthe molecular weight of the polyelectrolyte in the outer layer or byemploying monomers in the polyelectrolyte that provide mechanicalstrength, such as MMA.

The membrane can be formed by complex coacervation by combining drops ofa solution of biopolymer containing a cell suspension with a solution ofsynthetic polymer at physiological or neutral pHs of from about 6 toabout 8 so as to avoid adversely affecting the viability of the cells.In such process, the biopolymer is dissolved in a suitable aqueoussolvent that will not adversely affect the viable cells. Such solventsare well known and include buffered saline, culture medium and the like.Similarly, the synthetic polyelectrolyte is soluble in and dissolved ina suitable solvent that will not threaten the viability of the cells.Such solvents include aqueous solvents such as buffered saline, culturemedium and the like. The solvent used for the biopolymer does not needto be the same solvent used for the synthetic polymer. Mild agitation ofthe polyelectrolytic solution can be utilized if desired.

In one suitable technique, a substrate polymer solution containing acell suspension in a suitable diluent such as phosphate buffered saline(PBS) is added dropwise to a receiving solution containing syntheticpolyelectrolyte of the opposite charge in PBS at ambient temperature. Acohesive membrane is formed at the interface of the two solutions toprovide encapsulated cells. Advantageously, no organic solvent isrequired and no cross-linking reaction is necessary. Thus, theconditions of encapsulation are especially mild, yielding little cellmortality.

The proper matching of biopolymer and synthetic polyelectrolyte can bereadily confirmed. A drop of a solution of biopolymer can be added to asolution of electrolyte. A proper match results in the rapid formationof a microcapsule or membrane by complex coacervation, which can beobserved visually. The suitability of a given encapsulate regardingpermeability can be readily determined by in vitro tests using standardcell culture media to determine if desired products are secreted, ifunwanted immune components are excluded, and if viability ofencapsulated cells is suitably maintained.

The concentrations of the polymer solutions, the size of the dropletsadded to the synthetic polyelectrolyte solution, and the rate at whichthe substrate polymer solution containing cell suspension is added tothe synthetic polyelectrolyte solution can be adjusted to achieve anencapsulating membrane having the desired thickness of layers anddesired size. Suitable concentrations for the biopolymer solution andfor the synthetic polyelectrolyte solution will vary depending upon thespecific polymers and solvents employed, but determination of suchconcentrations is easily within the skill of the art. While it is notpossible to delineate concentrations for all possibilities, theconcentration of the biopolymer often will be from about 0.1 to 2%whereas the concentration of the synthetic polyelectrolyte often will befrom about 2 to 6%.

The thickness of the inner, substrate polymer layer, will depend on,inter alia, the viscosity of the biopolymer solution and the degree ofpenetration into the synthetic polyelectrolyte solution achieved by thesubstrate polymer solution droplets. The degree of penetration isrelated to the molecular weight of the polyions and the viscosity of thesolutions. The thickness of the outer shell may vary over a wide range,depending on the material used (e.g., whether the microcapsule isType-II, Type-III, or Type-IV as characterized herein) as well as theproperties desired for a particular application. The outer shell mostoften has a thickness of from about 1 to about 20 μm.

The practice of this invention provides microspheres that may range insize from as small as about 30 μm to as large as several millimeters.The larger sizes are most suitable for cells that tend to aggregate suchas islet of Langerhans cells and the like.

The number of cells within each microcapsule can be readily controlledand is a function of the density of the cell suspension within thebiopolymer. For example, cells in PBS (which may be at densities of 10³to 10⁶ cells per ml) can be mixed with the biopolymer to provide avariety of cell concentrations. Individual microcapsules can contain anydesired number of cells, typically ranging from 1 to 200 cells or more.Collagen gel has been observed to exhibit a “skin effect” that isdetrimental to mass transport, as a high concentration of collagen leadsto gelation. Such “skin effect” is concentration- andtemperature-dependent. Extra-cellular matrices like collagen or Matrigelhave gelling temperatures of ˜22-35° C. depending on the concentrationof these proteins. At 37° C., where hepatocytes are normally cultured ina bioreactor or transplantation is performed in vivo, the “skin effect”can be most pronounced. Since mass transport is among the most importantconsiderations for the design of bioreactors in BLAD, it is desirable toemploy the optimal concentration of collagen such that the “skin effect”is minimized while there still is enough collagen to complex with thesynthetic polyanion forming stable microcapsules.

Albumin was used as a model molecule for the permeability optimizationof the microcapsules. Albumin (MW ˜67,000 Da) is one of the secretedproteins of hepatocytes. It acts as a carrier to bind most metabolicwastes in the liver for removal from the blood. Another major scavengerprotein is bilirubin (˜10,000 Da), which is smaller than albumin.Albumin was found to be freely permeable to the microcapsules. A knownconcentration (1% w/v) of albumin was added to collagen andmicrocapsules were formed. The microcapsules were equilibrated in aculture medium with the same concentration of albumin (1% w/v) at 37° C.for 2 hours to allow a possible “skin effect” to occur. Suchequilibration before the permeability measurements is essential fordetecting any “skin effect” from the gelling collagen. Pre-equilibrationfor up to 5 days indicated that the “skin effect” was marginally morepronounced than with the 2 hour pre-equilibration. The albumin releasedfrom the microcapsules into the fresh culture medium with no albuminadded was thereafter monitored. With 1.5 mg/ml of the modified collagen,most of the encapsulated albumin was released from the microcapsuleswithin 15 minutes. As the concentration of collagen in the microcapsulewas increased to 4 mg/ml (˜0.4% w/v), the release of albumin was greatlyinhibited. For collagen concentration below 1.5 mg/ml, hepatocytes couldnot be encapsulated, possibly due to insufficient positive charge fromthe diluted collagen. Therefore, 1.5 mg/ml of modified collagen was usedfor all other experiments.

One preferred ter-polymer composition is made up of 25 mol % HEMA, 25mol % MAA and 50 mol % MMA at a concentration of 10% in PBS. When theter-polymer composition was modified for higher negative charge at theexpense of mechanical stability (e.g., 50 mol % MAA, 25 mol % HEMA, 25mol % MMA), the urea-synthesis of the encapsulated hepatocytes decreasesto levels below the monolayer control. The polymer composition andconcentrations can be varied to achieve enhanced mechanical stabilityand other physical characteristics.

Because the membrane of the encapsulated cells of the inventionprecludes contact between the cells and the host's immune mediators, alltypes of living cells, including both naturally-occurring andgenetically-engineered cells, may be encapsulated. The encapsulates aresuitable for anchorage-independent cells and are particularly suitablefor encapsulation of environmentally sensitive, anchorage-dependentliving cells such as hepatocytes.

Encapsulated cells of the invention also are useful as, for example, ahormone-producing system. Use of cells microencapsulated in aselectively permeable biopolymeric membrane affords the opportunity toprovide artificial organs and other methods for improving and restoringfunctions in people with physical disabilities.

An example of one type of hormone-producing cell is a cell of theanterior pituitary gland. Such cell excretes growth hormone, which interalia stimulates skeletal growth. In accordance with the invention,encapsulated naturally-occurring anterior pituitary cells are useful instimulating skeletal growth in a host. The encapsulated cells providegrowth hormone produced by the cells and introduced to the blood of ahost through the encapsulating membrane. Growth hormone also can beproduced by genetically-engineered microorganisms. Such microorganisms,when encapsulated, may be used to provide growth hormone to a host.

Encapsulated cells that secrete hormones also may be suspended in aculture medium and will excrete hormone over an extended period.Encapsulated insulin-producing cells, for example, mammalian pancreaticalpha cells, beta cells, or intact islets of Langerhans, may also beused as an artificial pancreas. Such encapsulated cells can be implantedinto a diabetic mammal and will function in vivo to excrete insulin andother hormones in response to host blood glucose concentration.

Other types of cells also may be beneficially encapsulated. For example,encapsulated neurotransmitter-secreting cells may be used to treatneurological disorders such as Parkinson's and Alzheimer's diseases.Similarly, chromaffin cell transplants may be used for alleviation ofpain, especially chronic pain, and encapsulated chondrocytes may be usedfor repair of musculoskeletal defects. Skilled practitioners recognizethe utility of encapsulating living cells, and will be able to identifystill further cells suitable for encapsulation in accordance with theinvention.

Even though the membrane may be permeable to proteases that can digestcollagen and other biopolymers used to form the inner layer of themembrane, it has been found that the inner layer remains intact. Withoutbeing bound by any theory, it is believed that the proteases cannotdigest the modified collagen, HA, modified HA, or other biopolymer whenthe biopolymer is complexed with the outer layer. This resistance can beanalogized to the resistance to solubilization of type I collagen and tocross-linked collagen, such as is found in heart valve tissue. Again,without wishing to be bound by theory, it is postulated that thecomplexation shields or changes the conformation of the cleavage site(between glycine and leucine), thus making the resulting complexedbiopolymer resistant to degradation.

The length of the period during which encapsulated cells remain intactwill depend upon the properties of the medium in which the encapsulatedcells are used and upon the composition of the biopolymer and of thesynthetic polyelectrolyte. For example, encapsulated cells used in aculture medium might be expected to remain intact for a longer periodthan encapsulated cells introduced into a human or animal body. Also,the mechanical stability of the membrane can be improved by increasingthe molecular weight of the synthetic polyelectrolyte. Skilledpractitioners will be able to determine the length of the period duringwhich encapsulated cells remain intact in various media.

Much effort on cell micro-encapsulation has focused on the materials andprocesses that make microcapsules for cell transplantation, cell-baseddrug delivery, and culture in bioreactors. There are a number ofconsiderations for making microcapsules, but some are more important forcertain applications than the others. For example, islet transplantationhas more stringent requirements on immune isolation thanmicroencapsulated hepatocyte cultured in an extra-corporeal bioreactor,which requires microcapsules with good mass transfer properties. This isbecause transplanted islets must function in vivo for extended periodsof time, while the latter is often used for a few hours ex vivo.However, there are characteristics such as the good microenvironment forcell viability and functions, and the maximal permeability for oxygenand nutrient supply, which are important for all applications. Thepresent invention provides a multi-layer microcapsule system based onsuch common characteristics, while allowing the other characteristics tobe imparted by the individual layers of the microcapsules.

The four types of microcapsules (Types I-IV) according to preferredembodiments of the invention all share the same inner layer of modifiedcollagen at 1.5 mg/ml. At such low concentration, the modified collagendoes not completely gel but surrounds the cells loosely in a semi-gelstate. Such a configuration provides a very good microenvironment forcellular functions. For the Type-II, -III, and -IV microcapsules, theadditional layers most preferably should minimize the permeabilityimpedance to the innermost layer to maintain good microenvironment foroptimal cellular functions.

The four types of microcapsules (Types I-IV) described herein have somedegrees of freedom for tuning the critical aspects of the microcapsuleperformance for different tissue engineering applications: goodmicroenvironment for cellular functions, mechanical stability, completeencapsulation and selective permeability for potentially more reliableimmune isolation. Type-I microcapsules provide a good microenvironmentfor cellular functions and exhibit good mass transfer properties, butare mechanically unstable and cannot ensure complete cell encapsulation.Type-I microcapsules are useful for applications such as the single-usebioreactor of an extra-corporeal device.

Type-II microcapsules have a more thorough cell encapsulation, whichmight provide a potentially more reliable immune isolation as well asbetter mechanical stability. Type-II microcapsules are more suitable forshort-term applications such as hepatocyte transplantation where thetransplanted hepatocytes are intended to stimulate the liverregeneration within a few weeks.

Type-III and -IV microcapsules provide significantly improved mechanicalstability. Mechanical stability can be especially improved by usingbiocompatible materials such as alumina to form a macro-porousexoskeleton outside Type-I microcapsules. Polymers that carry positivecharges, such as chitosan, can complex with the negatively chargedter-polymer shell. The micro-porosity of the exoskeleton layer can becontrolled by the concentration of chitosan. As alumina possesses arelatively high point of zero charge (PZC) of 9.0, the particles ofalumina sol at the physiological pH (7.2-7.4) possess positive charges.The positive charges on the alumina sol are neutralized by thenegatively charged ter-polymer at pH 7.4. Due to this chargeneutralization reaction, the alumina particles that originally repeleach other due to their mutual positive charges begin to condense,resulting in the formation of a stable, macro-porous alumina network(FIG. 2B). Such an alumina sol-gel exoskeleton can be formed andoptimized over a relatively wide range of concentrations. Type-IIImicrocapsules are mechanically very stable over a period of 7 days asmeasured by nano-indentation assay. These mechanically stablemicrocapsules are expected to be suitable for culturing cells in adynamic environment such as a fluidized bed bioreactor for large-scalecell culture or bio-artificial organs.

Type-IV microcapsules have an additional ter-polymer shell outside theexoskeleton of Type-III microcapsules. The negatively charged surfacesmay minimize the adsorption of plasma proteins. With the relativelystable exoskeleton covered by a selectively permeable, negativelycharged ter-polymer shell, the Type-IV microcapsules with two layers ofbuilt-in immune isolation features can allow plasma or even the wholeblood to directly contact the microcapsules without the use of a hollowfiber membrane for immune isolation. The Type-IV microcapsules areexpected to be useful in bio-artificial liver and in celltransplantation applications.

EXAMPLE

The following example is illustrative of preferred aspects of theinvention and should not be construed to limit the scope of the presentinvention. All reagents were purchased from Sigma-Aldrich unlessotherwise indicated.

Ter-Polymer Preparation

Ter-polymer of methacrylic acid (MAA), 2-hydroxyethyl methacrylate(HEMA), and methyl methacrylate (MMA) was synthesized by solutionpolymerization in 2-propanol=using 2,2′-azobisisobutyronitrile (AIBN) asinitiator. The monomers were distilled under nitrogen at reducedpressure. The polymerization was performed with an initiatorconcentration of 0.1 mol % of monomers under nitrogen with a magneticstirrer at 78° C. in an oil bath. The molar feed ratio of MAA, HEMA, andMMA was fixed at 25:25:50 or other ratios as desired and the ratio oftotal monomer to solvent at 1:6 (W/V). The reaction was allowed toproceed for overnight and quenched by cooling to room temperature. Thepolymer was precipitated by addition to a large excess of petroleumether. The precipitate was re-dissolved in a minimum volume of ethanol,and re-precipitated in distilled water. Recovered polymer then wasdissolved in a 1 M sodium hydroxide solution, and further purified byrepeated dialysis against distilled water with MWCO of 3500, andlyophilized. The yield of the polymer was found to be ˜63%. The polymercomposition was determined by proton NMR and the molar ratio of MAA,HEMA, and MMA was found to be 20.4:27.4:52.2 for the molar feed ratio of25:25:50. The molecular weight of the ter-polymer before dialysis wasdetermined by GPC (with THF as eluent) to be 30,000.

Modification of Collagen

Collagen can be modified to be cationic and anionic by the removal ofeither the negative or the positive charge from the collagen chains. Inthis case, cationic collagen was obtained through the modification ofthe carboxyl group by esterification with low molecular weight alcohol.20 ml of stock solution (3 mg/ml) of collagen (Vitrogen 100, CollagenCorp., Palo Alto, Calif.) was first precipitated with 400 ml of acetone.The precipitated collagen was dissolved in 200 ml of 0.1 M HClcontaining methanol (Merck), stirred at 4° C. for 6 days under sterileconditions. The lyophilized modified collagen can then be stored up to 6months in −20° C. in the presence of desiccant. The modification wasmonitored by titration. Titration of the natural collagen gave a typicaltitration curve of a mixed acid or a dibasic acid, while that ofmodified collagen gave a typical titration curve of a week monobasicacid, indicating that most carboxyl groups have been esterified inmodified collagen. In addition, neutralization of the modified collagenneeds less sodium hydroxide than that of the natural collagen,indicating that the polymer chain of the modified collagen has lessionic groups because of the esterification of the carboxyl groups.

Isolation of Hepatocytes

Hepatocytes were harvested from male, Wistar rat, weighing from 250-300g by a 2-step in situ collagenase perfusion. The rat was given 100 U/kgof heparin 30 minutes before anesthesia. Pentobarbital was administeredat a dose of 30 mg/kg, intra-peritoneally at the start of the operation.After laparotomy, a portal cannula was placed and fixed in a positionalong the portal vein. A cut was rapidly made in the lower vena cava. Inthe first 2-3 minutes, pre-perfusion (with Ca²⁺-free perfusion buffer)was performed while the liver remained in situ. The perfusate flow wasstarted at a rate of 50 ml per minute. While pre-perfusion was carriedout, the liver was transferred to a petri-dish and placed in a positionsimilar to its in situ site. After 10 minutes of pre-perfusion withCa²⁺-free medium, the liver was then perfused with recirculating 0.05%collagenase buffer for another 10 minutes. This was terminated when thevena cava ruptured. The entire perfusion procedure was performed underoxygenation that greatly improved the cell viability. The cells wereliberated from the connective vascular tissue and re-suspended in freshgrowth medium. This was followed by incubation of the cell suspension ina 37° C. CO₂ incubator for 30 minutes. The cell suspension was thenfiltered through a nylon mesh with a 60-μm pore size to further removethe connective tissue debris. The filtrate was then centrifuged at 50 gfor 1 minute to obtain the cell pellet. The cells were collected andwashed twice with growth medium. The viability of the hepatocytes wasdetermined to be 90-95% in all cases using the conventional Trypan Blueexclusion test.

Preparation of Exoskeleton Materials

A. Preparation of Alumina Sol

Aluminum sec-butoxide (7.5 ml) was slowly added to 250 ml deionizedwater at 85° C. The suspension of white precipitate was magneticallystirred for half an hour, after which 0.15 ml of fuming hydrochloricacid (37%) was added to the suspension and kept in a stoppered vessel at95-100° C. for 3 days. The resulting alumina sol was cooled to roomtemperature before use. The concentration of sol in term of aluminum(Al) concentration is 0.1168 M. The stock solution is further dilutedwith PBS to the desired working concentration (0.003 M to 0.006 M)before use.

B. Preparation of Chitosan

A stock solution of 2% chitosan (MW 400,000) was prepared by dissolvingin 0.5% acetic acid at 95° C. The final working concentration of0.01-0.02% was achieved through dilution of the stock with PBS.

Preparation of Microencapsulates

Micro-encapsulation was performed at room temperature with the aid of asyringe pump (IVAC P6000, Alaris Medical Systems, San Diego, Calif.).The microcapsules were incubated at 37° C. for one hour to allow themodified collagen to partially gel harvested by sedimentation andsubsequently washed twice with 1× Phosphate Buffered Saline (PBS) forfurther studies (Type-I microcapsules).

Type-II microcapsules were prepared by re-encapsulating the Type-Imicrocapsules in 1.5 mg/ml of modified collagen solution and 10%ter-polymer solution using the same method as in the Type-I microcapsulepreparation.

Type-III microcapsules were prepared by re-suspending the Type-Imicrocapsules in the respective exoskeleton materials for about 3minutes for the formation of a macro-porous network, followed byextensive washing in PBS before in vitro culture.

Type-IV microcapsules were prepared by re-encapsulating the Type-IIImicrocapsules in a 10% ter-polymer solution.

In Vitro Culture

The microcapsules were cultured for the required amount of time inHepatozym Serum free medium (SFM, GIBCO Laboratories, Chagrin Falls,Ohio) in a 35 mm polystyrene dish in a humidified atmosphere with 5%CO₂. The culture medium was supplemented with 10⁻⁷ M dexamethasone, 10nM insulin (Boehringer Mainnhem), 20 ng/ml epidermal growth factor and1% Penicillin and Streptomycin. After 1 day of culture, themicrocapsules were incubated in the medium with 1 mM of NH₄Cl for 90minutes before the medium was collected for urea assay. Themicrocapsules were then cultured in fresh medium again.

Functional Analysis of the Microencapsulated Hepatocytes

The samples collected at each time point were assayed for the ureaproduction calorimetrically with the Urea Nitrogen Diagnostic kit (SigmaDiagnostic). Data from three independent encapsulation experiments wereanalyzed and normalized against 10⁶ cells.

Permeability Assay for the Microcapsules

Bovine serum albumin (BSA) was suspended in 1.5 mg/ml of modifiedcollagen solution to reach a final concentration of 1% (w/v) andmicrocapsules of the four types (I-IV) were formed as previouslydescribed. The microcapsules were incubated at 37° C. for one hour forthe gelation of the collagen. After the incubation, the microcapsuleswere transferred to a 2 ml PBS solution containing equal concentrationof BSA, and allowed to equilibrate for 2 hours. The microcapsules werethen washed with PBS and the BSA release profile over a 2-hour intervalwas obtained. BSA level was determined using the Detergent Compatibility(DC) protein assay (Bio-Rad Laboratories). The percentage of releasedBSA in PBS was plotted over time with respect to the BSA standards.

Light Microscope Imaging of the Microcapsules

The microcapsules were visualized in an inverted microscope (OlympusCK40, Tokyo, Japan) with phase-contrast optics. The numbers ofhepatocytes within microcapsules were counted with the aid of ahemocytometer (Fuchs-Rosenthal).

An Olympus FLUOVIEW confocal microscope was used to image theter-polymer and the encapsulated hepatocytes in transmitted mode. Thethickness of the ter-polymer shell was measured with the softwareassociated with the Olympus FLUOVIEW confocal microscope.

Scanning Electron Microscopy (SEM)

The microcapsules were fixed with 3% glutaraldehye on a coverglasscoated with poly-L-lysine for an hour after which they were washedgently with 1×PBS for 5 minutes. The microcapsules were then post-fixedwith osmium tetra-oxide for 1 hour and dehydration was accomplishedusing a graded series of ethanol (25%, 50%, 75%, 95%, and 100%). Themicrocapsules were then critical point dried for about 2 hours inabsolute alcohol and mounted onto an aluminum stub and sputter coatedwith gold before viewing under a scanning electron microscope (Joel 5600LV).

Assessment of Mechanical Stability by Nano-Indentation

Indentation measurement was done using a UMIS-2000 Nano-indenter(Australian Scientific Instruments). The three-faced pyramid indentertip with an inclusion angle of 90° has well-defined geometry thatenables the quantitative measurements of the mechanical propertieslocated on the outermost shell or membrane of interest. The load anddepth of penetration were measured by two LVDT (Linear VariableDifferential Transformer) sensors independently. From the experimentallydetermined load-penetration data, hardness and modulus were determinedthrough the following analysis:H=P/AE/(1−ν²)=(π^(1/2))/2·A ^(−1/2) ·dP/dhwhere H is hardness of the specimen, P is indentation load, A is thetrue contact area at the maximum load, E is the elastic modulus of themicrocapsules, and ν is Poisson's ratio. dP/dh (called unloadingstiffness) essentially is the slope of unloading portion of theindentation load penetration data at the maximum indentation load.Average pressure that the microcapsules can withstand under a sharppoint can be defined by applied load divided by contact area. The areaof the indentation is therefore related to the depth of penetration, foran ideal sharp Berkovich indenter, is:A=24.56h ²

Microcapsules of various types were added onto a 13 mm coverglass coatedwith poly-L-lysine. The elastic modulus and hardness of themicrocapsules were determined in the nano-indentation experiments withthe maximum load of about 0.15 mN. This gave the penetration depth intothe polymer of slightly less than 2 pm before rupture at day 7 for theType-I microcapsule. The load was applied through a piezoelectricactuator in 15 steps to the maximum load. For each type of microcapsule,an average of 15 microcapsules were indented at the same load for eachexperiment. Data from three experiments were collected, with one indenton each microcapsule.

Results

Type-I microcapsules were developed with good microenvironment forenhanced cellular functions. Further development of other types ofmicrocapsules should also maintain such a good microenvironment. Toaccomplish this, a semi-gel-like inner collagen layer (1.5 mg/ml) shouldsurround the cells to provide a “loose” extra-cellular matrixconfiguration that mimics the in vivo situation. In Type-Imicrocapsules, the positively charged collagen layer was encapsulated bya thin layer (2-5 μm) of the negatively charged 10% ter-polymer shell.Other layers were added for specific applications with minimaladditional thickness to avoid adverse effects to the functions of themicroencapsulated cells.

Type II Microcapsules

Type-I microcapsules typically have a 2-5 μm thin shell, which does notensure complete cell encapsulation. Like other single-shellmicrocapsules, the Type-I microcapsules have occasionally been observedwith cells protruding out of the microcapsules (FIG. 1). To improve thereliability of immune isolation of the live cells within themicrocapsules that are required for many applications, a two-stepencapsulation method was employed. To ensure minimal impedance of themass transfer properties and the functions of the encapsulated cells,the flow rate in the syringe pump was optimized such that the desiredminimal thickness of the additional layers was achieved as measured bythe confocal imaging. These particular Type-II microcapsules have fourseparate layers (two ter-polymer shells spaced by the two layers of themodified collagen) with the additional layers covering the protrudingcells (FIG. 1) for a potentially more reliable immune isolation than theType-I microcapsules.

Type III Microcapsules

Type-I microcapsules can maintain structural integrity in static cellculture vessels for a few days. The mechanical stability of themicrocapsules requires further improvement for some applications, suchas the more dynamic environment in a bioreactor. Therefore, it would bedesirable to employ suitable materials that can form highly porous, butmechanically stable, layer(s) outside Type-I microcapsules. It isbelieved that such outer layer(s) could behave like an exoskeleton toconfer mechanical stability while imposing no or little impedance tomass transfer properties of the microcapsules. Ideally, such anexoskeleton should be formed by complex coacervation to avoid exposureof live cells to organic solvents. Therefore, the exoskeleton materialsshould have net positive charge to interact with the negatively chargedter-polymer shell of the Type-I microcapsules.

Alumina and chitosan were tested for the ability to form macro-porousexoskeleton as examined by SEM. Such microcapsules with macro-porousexoskeleton are referred to herein as Type-III microcapsules. Chitosancan form a macro-porous exoskeleton outside Type-I microcapsules whenused in a range of about 0.01-0.02% (w/v) (FIG. 2A). Lowerconcentrations of chitosan below this range were found not to affordsufficient material to cover the entire microcapsule surface. Higherconcentrations of chitosan above this range were found to possess somuch positive charges that the Type-I microcapsules disintegrated uponcontact. Alumina sol also can form a macro-porous exoskeleton outsideType-I microcapsules when used in a range of about 3-6 mM (FIG. 2B).

Type IV Microcapsules

As many plasma proteins, such as serum albumin, are negatively charged(with pI<7) under physiological pH, some applications involving contactwith blood would require a negatively charged outer-shell to minimizenon-specific protein adsorption onto the microcapsules. Since Type-IIImicrocapsules have a positively charged exoskeleton on the surface,another thin (2-5 μm) layer of the negatively charged ter-polymer shellcan be formed outside the exoskeleton by complex coacervation. TheseType-IV microcapsules have a selectively permeable, smooth andmicro-porous fourth layer (FIG. 3) to ensure complete cell encapsulationfor a potentially more reliable immune isolation than the Type-IIImicrocapsules.

Permeability Profile of the Microcapsules

For all applications, the microcapsules need to be permeable tonutrients, oxygen and metabolic wastes. Some applications requireselective permeability to allow nutrient exchange and at the same timeto prevent the passage of large molecules, such as immunoglobulins, intothe microcapsules. The permeability profiles of the Type-I, -II, -III,and -IV microcapsules were characterized to assess and optimize thesuitability of different types of microcapsules for use in relevantapplications. BSA with a MW of 66 kDa was encapsulated inside themicrocapsules and the rate of release from the microcapsules wasmeasured by spectrophotometry. Type-I microcapsules (∘) were used as acontrol since they release almost all the encapsulated BSA within 15minutes (FIG. 4). Type-II (□), Type-III (x), and Type-IV (⋄)microcapsules all exhibited some degrees of reduction in permeability.Within about 15 minutes, Type-II microcapsules (□) had about 80% of theBSA released, while Type-III (x) and Type-IV (⋄) microcapsules had about65% of the BSA released. By about 30-50 minutes, almost all theencapsulated BSA was released from all the microcapsules. Since Type-Imicrocapsules are impermeable to molecules larger than about 150 kDa,the other types of microcapsules should also be impermeable to largermolecules, such as immunoglobulins, which mediate the immune response.

Functional Profiles of the Microcapsules

As Type-II (□), Type-III (x), and Type-IV (⋄) microcapsules exhibitedsome degrees of permeability reduction (FIG. 4) when compared to Type-Imicrocapsules (∘), it is important to investigate the effects ofpermeability reduction on the functional status of the microencapsulatedcells. The urea production profiles of the microencapsulatedhepatocytes, which are very sensitive to the extra-cellularmicroenvironment, were characterized. All four types (I-IV) ofmicrocapsules were found to exhibit similar levels of hepatocytefunctions on day 1 (which is defined as 24 hours after the initial cellencapsulation) (FIG. 5). From day 1 onward, the urea production by theType-II (Δ), Type-III (x), and Type-IV (∘) microcapsules decreased oneafter another from the level exhibited by the Type-I (□) microcapsules.The urea production by the Type-II microcapsules (Δ) started decreasingfrom day 1 to about 40% of Type-I microcapsules (□) on day 3 and thenstabilized from day 3 onwards. The urea production by Type-IIImicrocapsules (x) started decreasing from day 4 to about 50% of Type-Imicrocapsules (□) on day 5. The urea production by Type IV microcapsules(∘) decreased from day 3 to about 45% of Type-I microcapsules (□) ondays 4-5. On days 6 and 7, all four types of the microcapsules exhibitsimilar levels of hepatocyte function, which is approximately theinitial level of the monolayer control (▪).

Mechanical Stability of the Microcapsules

Type-III and -IV microcapsules were developed to improve mechanicalstability. Nano-indentation with a pyramidal indenter tip with aninclusion angle of 90° was used to characterize the mechanical stabilityof the microcapsules over a period of 7 days (Table 1). Under the 0.15mN load, which is the maximum load at rupture for Type-I microcapsulesfor the pyramidal indenter, the depth of penetration into Type-Imicrocapsule shell increases rapidly from 0.4±0.1 μm on day 1 to 0.6±0.1μm on day 2 and 1.4˜0.8 μm on day 7. The depth of penetration of thepyramidal indenter tip into Type-II and Type-III microcapsule shellsremained relatively stable, in the range of 0.4±0.1 μm to 0.6±0.1 μm,throughout the 7-day period. There is a large variation in the depth ofpenetration of the pyramidal indenter on day 7, which was not observedon days 1 and 2, or in any other types of microcapsules. The protrudingcells not encapsulated by the ter-polymer shell might have contributedto such a large variation. The additional layers of either theter-polymer shell or the exoskeleton could then stabilize Type-II and-III microcapsules over the 7-day period as compared to Type-Imicrocapsules. TABLE 1 Depth of Penetration (μm ± standard error ofmeans) Type-I Type-II Type-III Days Microcapsules MicrocapsulesMicrocapsules 1 0.4 ± 0.1 0.4 ± 0.1 0.4 ± 0.1 2 0.6 ± 0.1 0.4 ± 0.1 0.6± 0.1 7 1.4 ± 0.8 0.5 ± 0.1 0.4 ± 0.1

It will be apparent to those skilled in the art that variousmodifications and variations can be made in the compositions and methodsof the present invention without departing from the spirit or scope ofthe invention. Thus, it is intended that the present invention cover themodifications and variations of this invention provided they come withinthe scope of the appended claims and their equivalents.

1. A microcapsule for culturing anchorage-dependent cells comprising aninner extracellular matrix surrounding the cells and an outer shell ofsynthetic polymer surrounding and supporting the extracellular matrix;wherein said microcapsule is permeable to nutrients necessary to sustainnormal metabolic functions of the cells and to toxins released by thecells; and wherein said outer shell has a thickness of from about 1 toabout 20 μm.
 2. The microcapsule of claim 1 wherein said innerextracellular matrix comprises a biopolymer inner layer and abiocompatible synthetic polyelectrolyte outer layer, wherein said innerlayer and said outer layer have charges sufficient to form a complex ofsaid biopolymer and said polyelectrolyte.
 3. The microcapsule of claim 2wherein said outer shell comprises (i) a biopolymer selected from thegroup consisting of cationic collagen modified to have a pKi of at leastabout 9, anionic esterified hyaluronic acid, anionic amine-modifiedhyaluronic acid, fibronectin, and laminin, and (ii) a biocompatiblesynthetic polyelectrolyte having an electrolytic charge opposite to thatof the biopolymer.
 4. The microcapsule of claim 3 wherein saidbiocompatible synthetic polyelectrolyte of said outer shell comprises anacrylate ter-polymer of methacrylic acid, hydroxyethyl methacrylate, andmethyl methacrylate.
 5. A microcapsule for culturing anchorage-dependentcells comprising an inner extracellular matrix surrounding the cells, amacro-porous exoskeleton surrounding and supporting the extracellularmatrix; and an outer shell of synthetic polymer surrounding themacro-porous exoskeleton; wherein said microcapsule is permeable tonutrients necessary to sustain normal metabolic functions of the cellsand to toxins released by the cells; and wherein said outer shell has athickness of from about 1 to about 20 μm.
 6. The microcapsule of claim 5wherein said inner extracellular matrix comprises a biopolymer innerlayer and a biocompatible synthetic polyelectrolyte outer layer, whereinsaid inner layer and said outer layer have charges sufficient to form acomplex of said biopolymer and said polyelectrolyte.
 7. The microcapsuleof claim 5 wherein said macro-porous exoskeleton comprises at least oneof alumina, alumina sol, and chitosan.
 8. The microcapsule of claim 5wherein said synthetic polymer of said outer shell comprises an acrylateter-polymer of methacrylic acid, hydroxyethyl methacrylate, and methylmethacrylate.
 9. A method of preparing a microcapsule havinganchorage-dependent cells surrounded by an inner extracellular matrixand an outer shell of synthetic polymer surrounding and supporting theextracellular matrix, the process comprising: preparing an innerextracellular matrix having an inner layer and an outer layer,comprising extruding an inner layer biopolymer solution containingbioactive cells into a biocompatible synthetic polyelectrolyte outerlayer; wherein said inner layer and said outer layer have chargessufficient to form a complex of said biopolymer and saidpolyelectrolyte; and forming an outer shell by encapsulating said innerextracellular matrix containing said cells with a synthetic polymersolution, wherein said outer shell thus-formed has a thickness of fromabout 1 to about 20 μm.
 10. The method of claim 9 wherein said syntheticpolymer solution of said outer shell comprises (i) a biopolymer selectedfrom the group consisting of cationic collagen modified to have a pKi ofat least about 9, anionic esterified hyaluronic acid, anionicamine-modified hyaluronic acid, fibronectin, and laminin, and (ii) abiocompatible synthetic polyelectrolyte having an electrolytic chargeopposite to that of the biopolymer.
 11. The method of claim 10 whereinsaid biocompatible synthetic polyelectrolyte of said outer shellcomprises an acrylate ter-polymer of methacrylic acid, hydroxyethylmethacrylate, and methyl methacrylate.
 12. A method of preparing amicrocapsule having anchorage-dependent cells surrounded by an innerextracellular matrix and a macro-porous exoskeleton surrounding andsupporting the extracellular matrix, the process comprising: preparingan inner extracellular matrix having an inner layer and an outer layer,comprising extruding an inner layer biopolymer solution containingbioactive cells into a biocompatible synthetic polyelectrolyte outerlayer; wherein said inner layer and said outer layer have chargessufficient to form a complex of said biopolymer and saidpolyelectrolyte; and suspending said inner extracellular matrixcontaining said cells in an exoskeleton material having a chargeopposite to that of the outer layer of said extracellular matrix to forma macro-porous exoskeleton over said extracellular matrix.
 13. Themethod of claim 12 wherein said macro-porous exoskeleton comprises atleast one of alumina, alumina sol, and chitosan.
 14. The method of claim12 further comprising forming an outer shell by encapsulating themicrocapsule in a synthetic polymer solution.
 15. The method of claim 14wherein said synthetic polymer solution comprises an acrylateter-polymer of methacrylic acid, hydroxyethyl methacrylate, and methylmethacrylate.
 16. A method of culturing anchorage-dependent cellscomprising applying agitation to the microcapsule of claim 1 after apredetermined time to rupture the outer shell, and removing theextracellular matrix to recover the cells.
 17. A multi-layermicrocapsule comprising bioactive cells attached to a microcapsulemembrane; wherein said microcapsule membrane comprises (i) a first innerlayer of biopolymer selected from the group consisting of cationiccollagen, anionic collagen, anionic esterified hyaluronic acid, anionicamine-modified hyaluronic acid, fibronectin, and laminin; (ii) a secondintermediate layer of polyelectrolyte synthetic polymer; and (iii) athird outer layer forming an exoskeleton to provide mechanicalstability; wherein said first inner layer and said second intermediatelayer are complexed via ionic charges; wherein said second intermediatelayer and said third outer layer are complexed via ionic charges;wherein said microcapsule membrane is permeable to molecules smallerthan or equal to the size of albumin, to nutrients necessary to sustainnormal metabolic functions of the bioactive cells, and to toxinsreleased by the bioactive cells; and wherein said microcapsule membraneis impermeable to immunoglobulins and macrophages.
 18. The multi-layermicrocapsule of claim 17 further comprising (iv) a fourth outer layercomprising a polyelectrolyte synthetic polymer surrounding said thirdlayer, wherein said fourth outer layer is complexed with said thirdlayer via ionic charges.
 19. The multi-layer microcapsule of claim 17wherein said second intermediate layer of polyelectrolyte syntheticpolymer is an acrylate ter-polymer of methacrylic acid, hydroxyethylmethacrylate, and methyl methacrylate.
 20. The multi-layer microcapsuleof claim 17 wherein said third outer layer comprises a material selectedfrom the group consisting of alumina, alumina sol, and chitosan.
 21. Themulti-layer microcapsule of claim 17 wherein said bioactive cellscomprise a mixture of dividing cells and non-dividing cells.
 22. Themulti-layer microcapsule of claim 21 wherein said bioactive cellscomprise a mixture of hepatocyte cells and non-parenchymal cells. 23.The multi-layer microcapsule of claim 4 wherein said third layercomprises a ceramic sol modified to be negatively charged, wherein saidthird layer is unstable at a physiological pH of 7.4 to provide ashort-term controlled release of cells, cell aggregates, or tissuestructures.
 24. A process of preparing a bioartificial liver assistdevice comprising packing one or more of the biocompatible microcapsuleof claim 1 in a bioreactor.
 25. A process of preparing a bioartificialliver assist device comprising packing one or more of the biocompatiblemicrocapsule of claim 5 in a bioreactor.
 26. A bioartificial liverassist device comprising one or more of the biocompatible microcapsuleof claim 1 contained within a bioreactor.
 27. A bioartificial liverassist device comprising one or more of the biocompatible microcapsuleof claim 5 contained within a bioreactor.
 28. A method of preparingliving cells for multi-dimensional imaging to study cells, tissue, ortissue constructs, the method comprising culturing at least one cell inthe microcapsule of claim 1 and imaging the cell using microscopy.
 29. Amethod of preparing living cells for transplantation comprisingculturing at least one cell in the microcapsule of claim 1, harvestingthe cell, and coupling the cell to a scaffold.
 30. A method of analyzingcells comprising removing at least one cell from a biopsy sample,culturing the cell in the microcapsule of claim 1, and performingcytometry analysis.